Method for producing branched fatty acids using genetically modified plants

ABSTRACT

The invention relates to plant cells comprising a recombinant nucleic acid coding for a product which induces or stimulates the synthesis of branched fatty acids in said cells. It also relates to a production process for branched fatty acids using these plant cells or plants containing it.

INTERODUCTION

[0001] The present invention relates to a production process for fattyacids. It relates more particularly to a production process for fattyacids with a non-linear aliphatic chain. The process of the inventionconsists more particularly in the use of genetically modified plants.The fatty acids thus produced, in a free or bound form, possiblymodified chemically or enzymatically, may be used in the manufacture ofvarious industrial products, in particular lubricants of the motor oilor hydraulic oil type.

[0002] The fatty acids are acids with an aliphatic chain of a lengthusually included betweeen 6 and 25 carbon atoms. The aliphatic chain maybe linear or non-linear (also designated branched). The fatty acidsproduced naturally exist in either the free or an esterified form, inparticular in the form of diglycerides or triglycerides. The fatty acidsare usually used in various types of industries, in particular for thepreparation of lubricant bases of natural origin or in detergents,adjuvants or even in the constitution of biocarburants. For all of theseapplications, it is particularly advantageous to be able to use fattyacids of natural origin, in particular derived from plant material.Thus, different production and extraction processes for fatty acids fromplants (in particular from plant seeds) have been described in the priorart (see in particular Harrington et al., Ind. Eng. Chem. Prod. Res.Dev. 1985, vol. 24, page 314; FR 2 722 798). Moreover, the applicationWO 94/13814 relates to plant cells genetically modified by an enzymeimplicated in the transfer of free fatty acids to cholesterol.Nonetheless, although this application allows the composition of thetriglycerides to be modified, it in no way allows the structure of thealiphatic chain of the fatty acids to be altered. Schmid K. (Plant LipidMetabolism (1995, 108) mentions the synthesis of cyclic fatty acids(dihydrosterculate) in genetically modified tobacco cells. However, thisdocument does not described the production of branched fatty acids, orcorresponding means or industrial applications.

[0003] One of the major disadvantages of the earlier processes usingplants consists precisely in the low diversity of the fatty acids whichmay be obtained. Thus, basically, the plants naturally produce fattyacids with a linear aliphatic chain of the C6 to C24 type. Now, thestudies conducted by the applicant Company have shown that to obtain agood lubricant base, it is particularly advantageous to use fatty acidswith a non-linear (branched) aliphatic chain. Thus the applicant hasshown that the use of (free or bound) fatty acids with a branchedaliphatic chain led to products of the lubricant type being obtainedwhich exhibit good thermal stability, good resistance to oxidation, goodviscosity, good biodegradability as well as low melting points (or pourpoints).

[0004] There thus exists a real need to have available processes makingit possible to produce branched fatty acids efficaciously. The presentinvention provides an advantageous solution to precisely these needs bydescribing processes based on the genetic modification of plants.

[0005] Thus, a first feature of the invention relates to a productionprocess for branched fatty acids by means of genetically modifiedplants.

[0006] Another feature of the invention lies in genetically modifiedplants capable of producing branched fatty acids and in a productionprocess for such plants. Another feature of the invention also consistsof plant cells containing a recombinant nucleic acid coding for aproduct which induces or stimulates the synthesis of branched fattyacids in said cell.

[0007] The present invention also relates to the recombinant nucleicacid such as defined above and to any vector containing it.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention thus relates to a production process forbranched fatty acids from genetically modified plants, to plants andcells of genetically modified plants producing branched fatty acids aswell as to recombinant nucleic acids and vectors containing them, whichcan be used for this purpose.

[0009] In the sense of the invention branched fatty acid means any fattyacid possessing a true branched aliphatic chain. As emerges from thetext of the present application, the term “branched” designates in thesense of the invention fatty acids bearing one or more substitutions atone or more distinct positions of the aliphatic chain. Thus, the solepresence of a ring in the aliphatic chain is insufficient to qualify thefatty acid as branched according to the invention. It is preferably afatty acid possessing a C6 to C24 aliphatic chain, and even morepreferably a C12 to C24 aliphatic chain. Examples of such acids,essentially in a saturated form and not yet branched, are in particularcapric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid(C16), stearic acid (C18), eicosanoic acid (C20), docosanoic acid (C22),tetracosanoic acid (C24) and mixtures of these latter. The aliphaticchain of the branched fatty acids of the invention may be branched atone or more positions by different groups. Advantageously, the aliphaticchain bears one to four branches located in the central part of thealiphatic chain and/or in its terminal part opposite to the acidfunction. More preferably, the branch(es) is (are) located on one ormore carbons included between the positions 8 and 15 of the aliphaticchain. Moreover, the branched group may be any alkyl group, preferably aC1 to C5 alkyl, still more preferably methyl, ethyl, propyl, isopropyl,butyl, tertbutyl. A particularly preferred branched group is a methylgroup. Particular branched fatty acids in the sense of the inventionare, for example, 9-methyl octadecanoic acid; 10-methyl octadecanoicacid; 9,12-dimethyl octadecanoic acid; 10,12-dimethyl octadecanoic acid;9,13-dimethyl octadecanoic acid; 10,13-dimethyl octadecanoic acid;9,12-dimethyl oleic acid; 10,12-dimethyl oleic acid; 9,13-dimethyl oleicacid; 10,13-dimethyl oleic acid or even 2,4,6,8-tetramethyl decanoicacid.

[0010] Moreover, the branched fatty acids according to the invention maybe branched fatty acids in the free form or in the form of derivatives.They may be in particular branched fatty acid esters, such asmonoesters, diesters or triesters of glycerol (triglyceride). In thecase of di- or triglycerides, it is moreover not necessary that all ofthe fatty acids present in the molecule be branched. Thus, atriglyceride may contain 1, 2 or 3 branched fatty acids. It is, however,preferable that the proportion of branched fatty acids in the esters orother derivatives be high.

[0011] A first object of the invention relates more particularly to aproduction process for branched fatty acids, characterized in that saidbranched fatty acids are produced from at least one plant cell or fromone plant material or from a plant comprising at least one cell, saidplant cell comprising in its genome a recombinant nucleic acid codingfor a product inducing or stimulating the synthesis of branched fattyacid(s).

[0012] In the sense of the invention “recombinant nucleic acid”designates any ribonucleic or deoxyribonucleic acid constructed bygenetic engineering techniques. It may be in particular a complementaryDNA, a genomic DNA or a messenger RNA. Such a nucleic acid may be ofvaried origin, in particular bacterial, viral, plant, mammalian,synthetic or semi-synthetic. A recombinant nucleic acid may be preparedby using the procedures known to the specialist skilled in the art, andin particular by screening of nucleic acid libraries with suitableprobes, by chemical synthesis (by using nucleic acid synthesizers), byenzymatic digestions/ligations, etc or by any combination of thesemethods.

[0013] The present invention may be used with different types of plants,preferably plants cultivated in fields. Advantageously, it is anoleaginous plant, such as for example colza, sunflower, peanut, soya,flax or even maize. It may also be a tobacco plant.

[0014] The plant cells according to the invention may be the cells ofany of the plants such as defined above. It may more preferably be acultivatable plant cell and capable of regenerating a plant.

[0015] The term “plant material” designates any part of a plant such asdefined above, comprising at least one cell. It may be any tissue ofsaid plant such as, in particular, seeds.

[0016] The cell or plant according to the invention is thus geneticallymodified, so that it comprises in its genome, in a free or integratedform, a recombinant nucleic acid coding for a product inducing orstimulating the synthesis of a branched fatty acid. The plants and plantcells of the invention thus comprise fatty acids, a fraction of which isconstituted by branched fatty acids. Depending on the applicationsenvisaged, it may be advantageous for this fraction to represent asubstantial proportion of the total fatty acids. Advantageously, therecombinant nucleic acid is present in the cell in a form integratedinto the genome of said cell, so as to be segregationally more stable.

[0017] A product is designated as inducing the synthesis of a branchedfatty acid when it allows the plant cell containing it to accomplish asynthesis which it could not have done in its absence. One speaks of aproduct stimulating the synthesis of a branched fatty acid when it is aproduct which allows the plant cell containing it to promote apreexisting synthetic pathway in said cell but one exploited little ornot at all by the latter, for example, for reasons of substratestoichiometry or specificity. The recombinant nucleic acid used in thepresent invention may code more particularly for a product capable ofinducing a branching of the fatty acids produced by the cellpost-synthesis or for a product enabling a branching concomitant withsaid synthesis to be achieved. Moreover, these two approaches may becombined.

[0018] The fatty acids are synthesized in the chloroplasts of the plantcells. This synthesis comprises several repetitive cycles during whichcarbon units are added successively to each other to generate the fattyaliphatic chain. After being synthesized these linear fatty acids areexported to the cytoplasm where they undergo various post-synthetiic (orpost-elongation) modifications and, in particular, a desaturation byenzymes designated desaturases.

[0019] In a first embodiment of the invention, the recombinant nucleicacid codes for a product making it possible to induce or stimulate thepost-synthetic branching of the fatty acids produced by said plant cell.More particularly, according to this first embodiment of the invention,the recombinant nucleic acid codes for an enzyme permitting the transferof one or more alkyl groups to the double bonds of the unsaturated fattyacids.

[0020] The alkyl groups transferred may be, as indicated above, anyalkyl group preferably comprising from 1 to 5 carbon atoms. They areadvantageously alkyl groups comprising from 1 to 3 carbon atoms and inparticular methyl, ethyl, propyl or isopropyl.

[0021] Advantageously, the recombinant nucleic acid codes for aheterologous enzyme, i.e. an enzyme derived from an organism other thana plant cell. More particularlly, the enzyme used is advantageouslyderived from a prokaryotic or eukaryotic microorganism such as, forexample, a bacterium or a yeast.

[0022] As an illustration of the recombinant nucleic acids according tothe invention, mention may be made of a recombinant nucleic acid codingfor cyclopropane fatty acid synthase, also designated CFAS. The nucleicacid may also code for methyl transferases such as, for example,SAM-methyl transferase, i.e. enzymes capable of catalyzing the transferof a methyl group from SAM (S-adenosyl-methionine) to an unsaturatedfatty acid or, more generally, to a doouble bond of an aliphatic chain.A particular recombinant nucleic acid in the sense of the inventioncodes, for example, for the cyclopropane fatty acid synthase such asdescribed by Wang et al. (Biochemistry 31 (1992) 11020) or any enzymeanalogous to this latter. The term “analogous” in the sense of thepresent invention means any enzyme possessing one or more structuralmodifications compared to the enzyme describe above and conserving analkyl group transfer activity towards the double bonds of an aliphaticchain. These structural modifications may be mutations, deletions,substitutions or additions of one or more amino acids. The term“analogous” also comprises the homologous sequences obtained from otherorganisms. Another “analogue” is an enzyme produced by a nucleic acidhybridizing with all or part of the sequence of the transferase orsynthase and conserving an activity of the same kind. The hybridizationis advantageously performed under conditions of normal or, preferably,high stringency. Stringent hybridization conditions are, for example:hybridization at 42° C., 50% formamide, 5× SSC, 1× Denhardt; washing at65° C. with 0.1× SSC, 0.1% SDS. Non-stringent hybridization conditionsare, for example: hybridization at 37° C., 40% formamide, 5× SSC, 1×Denhardt; washing at 50° C. with 1× SSC, 0.1% SDS. The stringentconditions are particularly suited when the test nucleic acid is presentin low amounts and/or in an only slightly purified form. Thenon-stringent conditions are more suitable when the test nucleic acid ispresent in larger amounts and in a form significantly represented in thesample. Other hybridization conditions are well known to the specialistskilled in the art (see, in particular, Maniatis et al.) Anotherparticular example of recombinant nucleic acids according to theinvention is a recombinant nucleic acid coding for the methyltransferase responsible for the synthesis of 9-methyl-10-hexadecenoicacid in Corynebacterium spp. (Niepel et al., J. Bact. 180 (1998) 4650)or in a variety of alga (Carballeira et al., Lipids 32 (1997) 1271).

[0023] Moreover, this embodiment consisting of transferring an alkylgroup to an aliphatic chain requires that alkyl groups or alkyl donorsare present in said cell. In this respect, the cell reserves for methylgroup transfer are constituted principally by the SAM group.Consequently, for the purpose of improving still further the efficacy ofthis reaction, and hence of the yield of branched fatty acids, aparticular embodiment consists of introducing into the plant cell asecond nucleic acid coding for an enzyme implicated in the synthesis ofthe alkyl donor, in addition to the nucleic acid coding for the productdefined above. Preferably, it is a nucleic acid coding for an enzymeinvolved in the synthesis of SAM. Even more preferably, it is a nucleicacid coding for SAM synthetase or an analogue of the latter. Thisnucleic acid may be introduced by the intermediary of a secondrecombinant nucleic acid, simultaneously or sequentially with respect tothe first. According to another embodiment, the nucleic acids coding forthe two enzymes are borne by the same recombinant nucleic acid.

[0024] According to a second embodiment of the present invention, therecombinant nucleic acid contained in the plant cell codes for a productmaking it possible to induce or stimulate the incorporation into thealiphatic chain of a fatty acid during its elongation groups generatingone or more branches. Accordingly, this second approach does not involvea post-synthetic modification of the fatty acids produced but achievesbranching concomitantly with said synthesis.

[0025] This embodiment is preferably obtained by forcing the plant cellto use as substrate for the synthesis of the aliphatic chain an acyl-CoAgroup bearing more than 3, and preferably between 4 and 8, carbon atomsinstead of malonyl CoA. Advantageously, the substrate used is anon-linear acyl-CoA group. Even more preferably, it is a non-linearacyl-CoA group permitting the incorporation of an odd number of carbonatoms into the aliphatic chain during its elongation. This strategyenables branched fatty acids called multibranched fatty acids to beobtained, such as for example 2,4,6,8-tetramethyldecanoic acid. Aparticular example of an acyl-CoA type substrate is for examplemethylmalonyl CoA. Methylmalonyl CoA is a non-linear group, bearing 4carbon atoms, three of which serve for elongation. In this case, for thepurpose of forcing the plant to use this substrate for the synthesis ofthe aliphatic chain, it is particularly advantageous to introduce intothe plant cell a recombinant nucleic acid coding for a malonyl CoAdecarboxylase (E.C. No. 4.1.1.9). The mode of action of this enzyme aswell as in a more general manner the synthetic pathway of the branchedfatty acids by the cells modified according to this strategy is shown inFIG. 1. It is understood that any other enzyme analogous to thatmentioned above and capable of forcing the plant to use an acyl-CoAbearing more than 3, and preferably 4 to 8, carbon atoms as substrate inthe synthesis of the aliphatic chains of the fatty acids may be used inthe present invention. As a particular example of such enzymes, mentionmay be made in particular of the malonyl CoA decarboxylase, thenucleotide sequence of which has been described in the literature (cf.the examples) as well as any analogue of the latter.

[0026] This second embodiment may also be obtained by modifying thespecificity of the enzymes implicated in the synthesis of the fattyaliphatic chain, in particular by modifying the specificity of theenzyme which prevents the transfer of malonyl CoA to malonyl ACP, and inparticular the specificity of all or part of the fatty acid synthase,acetyl CoA carboxylase or acetyl CoA transacylase. In this respect, thefatty acid synthase consists of a multi-enzyme complex and themodification of its specificity may be obtained by modification of one,several or even all of the subunits.

[0027] Moreover, as indicated above, these two embodiments may becombined to generate plant cells capable of synthesizing fatty acidswith a branched aliphatic chain both during and after elongation.

[0028] For the implementation of the present invention, the recombinantnucleic acid advantageously comprises regulatory regions of the promotertype which are functional in plant cells and which enable the expressionof the nucleic acids defined above to be regulated. Among the regulatoryregions of transcription which can be used in plants and in theimplementation of the present invention, mention may be made for exampleof the regions associated with the cauliflower mosaic virus (35S, 19S)or the promoter regions of structural genes such as the genes fornopaline synthase (nos) or octopine synthase (ocp) or mannopine,agropine or acyl carrier protein (ACP). The structure and cloning ofthese different regions have been described in the literature. Moregenerally, any promoter permitting constitutive, spatial or temporalexpression of a nucleic acid in a plant cell may be used for theimplementation of the present invention. In this respect, it is possiblein particular to use promoters which permit expression localized tocertain tissues or parts of the plant (seed, pollen, etc.), or promoterswhose activity depends on the stage of development of the plant(elongation, flowering, etc.). As a promoter which is expressedpredominantly in the seeds, mention may be made, for example, of the ACTpromoters of ACP and napine or the promoters described by Krebbers etal. (Plant Physiol. 87 (1988) 859). The use of such promoters mayprevent any potential side effect induced by a generalized and massiveproduction of branched fatty acids in the plant, and also facilitate therecovery of the fatty acids thus produced, by concentrating them incertain regions of the plant.

[0029] Moreover, it is also advantageous to introduce into therecombinant nucleic acids according to the invention terminatingsequences localized at 3′ which may derive either directly from the geneused or arise from other gene regions such as those in particular ofnopaline synthase (nos) or also octopine synthase (ocp), mannopine,agropine or ACP.

[0030] The recombinant nucleic acid which thus advantageously comprisesa regulatory region of transcription, a gene or region coding for anactive product such as defined above and 3′ terminating regions isadvantageously introduced into a plasmid cloning or expression vector.

[0031] In this respect, another object of the invention relates moreparticularly to a recombinant nucleic acid comprising:

[0032] a nucleic acid coding for a product such as defined above,

[0033] a promoter regulating the expression of said nucleic acid andcapable of causing the localized expression of this nucleic acid incertain plant tissues or certain plant parts, and

[0034] a 3′ transcription termination region.

[0035] More particularly, the promoter comprised in the geneticconstructions of the invention is a promoter capable of causing theexpression of the nucleic acid localized in the seed of the plant. Theterm “localized expression” signifies that the expression is markedlygreater in the tissue concerned than in the other plant tissues.Nonetheless, it is understood that an absolute specificty of expressionis not required, provided that a significant expression is observed inthe tissues selected. The localized character of expression according tothe invention is doubly advantageous because it facilitates, on the onehand, the recovery of the branched fatty acids and because it limits therisks of toxicity (in particular on good growth and reproduction) of theproducts expressed in the modified plants.

[0036] The recombinant nucleic acids of the invention may, moreover,comprise address signals which lead to improvement in addressing theproteins synthesized to the cell compartments where they exert theiractivity. When the product synthesized is a product implicated in thetransfer of alkyl groups to fatty chains post-elongation, the proteinexerts its activity in the cytoplasm and thus does not require specialaddress signals. When the product is implicated in the synthesis of thefatty chains itself, it exerts its activity within the chloroplasts andit may be advantageous to use a signal which addresses this product tothe chloroplast. Such signals are known in the prior art.

[0037] Another object of the invention relates to a recombinant nucleicacid comprising:

[0038] a nucleic acid coding for a methyl transferase capable ofcatalyzing the transfer of a methyl group to an aliphatic chain of anunsaturated fatty acid,

[0039] a functional promoter in the plant cells regulating theexpression of said nucleic acid, and

[0040] a 3′ transcription termination region.

[0041] In another particular embodiment, the object of the invention isa recombinant nucleic acid comprising:

[0042] a nucleic acid coding for a malonyl CoA decarboxylase,

[0043] a functional promoter in the plant cells regulating theexpression of said nucleic acid, and,

[0044] a 3′ transcription termination region.

[0045] Another object of the present invention also relates to a vectorcomprising a recombinant nucleic acid such as defined above. Such avector may be in particular a derivative of the vector Ti. The vectorsof the invention may be used to produce large quantities of recombinantnucleic acids in the cells or to produce the corresponding enzymes or totransform plant cells.

[0046] The present invention also relates to any plant cell comprising arecombinant nucleic acid or a vector such as defined above.

[0047] The recombinant nucleic acids or vectors of the invention may beintroduced into the plants by any technique known to the specialistskilled in the art, and in particular by chemical, physical orbiological procedures. The chemical techniques implicate for example theuse of transfecting agents which facilitate cellular penetration. Thephysical techniques are, for example, electroporation, bombardment,“gene guns”, etc. A well-known and particularly efficacious biologicaltechnique consists of using infiltrations under vacuum or co-culture,viral vectors and, preferably, Agrobacterium tumefaciens which enablesgenetic material to be introduced into the plant cell by means of theplasmid Ti. These techniques are well-known to the specialist skilled inthe art. After transformation, the plant cells actually containing thenucleic acid according to the invention are selected. The cells may thenbe maintained in culture, possibly multiplied in culture and useddirectly for the production of branched fatty acids (batch cultures, fedbatch, etc.). These cells may also be stored for subsequent use. Thesecells may also be used to regenerate transgenic plants, according tostandard procedures used in plant biology.

[0048] Thus, another object of the invention relates to a process forthe preparation of a transgenic plant capable of producing branchedfatty acids, comprising the introduction of a recombinant nucleic acidsuch as defined above into a plant cell or plant part and theregeneration of a transgenic plant from these plant cells or plantparts. Advantageously, the introduction is achieved by usingAgrobacterium. In a particular embodiment, the introduction is made intoplant cells or foliar disks. The introduction is performed by anytechnique known to the specialist skilled in the art.

[0049] Another object of the invention relates to a transgenic plant,the cells of which contain a recombinant nucleic acid such as describedabove and which codes for the enzymes permitting the post-syntheticbranching of the fatty acids produced by said plant cell to be inducedor stimulated, or sequences inducing or stimulating the incorporation ofbranched groups into the aliphatic chain of a fatty acid duringelongation.

[0050] The transgenic plants of the invention contain the recombinantnucleic aid stably integrated into the genome of both germ cells andsomatic cells.

[0051] The invention also relates to any plant material derived fromsuch a plant, and in particular to the seeds.

[0052] The invention also relates to a production process for branchedfatty acids by culture of a cell such as defined above and recovery ofthe fatty acids produced.

[0053] The invention also relates to a production process for branchedfatty acids by cell culture of a plant cell comprising:

[0054] the culture of these cells in a medium suitable for their growth,

[0055] the extraction and purification of the branched fatty acids fromthe cell biomass, and in particular from these cells or from thesupernatant of said culture.

[0056] The invention also relates to a preparation process for branchedfatty acids by extraction from a transgenic plant, the cells of whichcontain a recombinant nucleic acid such as defined above according towhich said fatty acids are recovered from any part of said plant, and inparticular from the seeds of said plant.

[0057] The invention relates to a preparation process for branched fattyacids from a transgenic plant, the cells of which contain a recombinantnucleic acid comprising:

[0058] the field culture of said transgenic plants;

[0059] the recovery of the seeds of said plants;

[0060] the extraction of the fatty acids from these seeds.

[0061] The process of the invention is quite especially suited to theproduction of non-cyclic methylated fatty acids, in particular C16 orC18 fatty acids bearing one or more methyl groups. As preferred examplesmention may be made of 9-methyl octadecanoic acid, 10-methyloctadecanoic acid, 9-methyl hexadecanoic acid and 10-methyl hexadecanoicacid.

[0062] In the production processes for branched fatty acids such asdefined above, an optional treatment step of the fatty acids may beperformed in order to improve still further the physicochemicalproperties of these compounds. Thus, the processes of the inventioncomprise in particular:

[0063] an extraction step of the branched fatty acids from the cells,seeds or plants, and,

[0064] a treatment step of the fatty acids to improve theirphysicochemical properties.

[0065] The extraction may be performed by the standard methods known tothe specialist skilled in the art mentioned above.

[0066] More preferably, the treatment step is a hydrogenation step,performed under standard or forcing conditions, leading to the formationof saturated branched fatty acids. More specifically, the hydrogenationmay be performed under the conditions described by Christie W. W.(Topics in Lipid Chemistry, 1970, vol. 1, F. D. Gunstone, ed. London:Logus Press)—incorporated into the present description by reference—,optionally in the presence of a palladium catalyst.

[0067] More generally, the invention relates to the use of a transgenicplant regenerated from a cell such as defined above for the productionof branched fatty acids.

[0068] The invention also relates to the use of a plant or part of atransgenic plant, at least some of the cells of which contain arecombinant nucleic acid such as defined above for the production ofbranched fatty acids.

[0069] Finally, the invention relates to branched fatty acids capable ofbeing obtained by the process such as that described above. Theinventors have shown in particular that the branched fatty acid esterssuch as defined in the invention have good resistance to oxidation andlow pour points, and thus possess advantageous properties for use aslubricants.

[0070] Hence, the invention relates to the use of branched fatty acidesters such as defined above as lubricants, in particular for thepreparation of oil compositions, in particular motor oil and hydraulicoil, in particular by replacing unconventional or synthetic bases (suchas esters, polyalphaollefins, etc.).

[0071] The invention also relates to a lubricantt composition comprisingbranched fatty acids such as described above and one or more additivessuch as in particular anti-wear agents, pressure modifiers, surfactants,etc.

[0072] The present application will be described in more detail with theaid of the examples which follow, which must be considered asillustrative and non-limiting.

LEGENDS TO FIGURES

[0073]FIG. 1: Schema of the synthesis of fatty acids in the chloroplastof the plant cell.

[0074]FIG. 2: Representation of a recombinant nucleic acid comprising agene coding for a transferase under the control of the promoter CaMV 35S(FIG. 2A) or nos (FIG. 2B).

[0075]FIG. 3: Representation of a recombinant nucleic acid comprising agene coding for SAM synthase.

[0076]FIG. 4: Restriction map of the cfa gene.

[0077]FIG. 5: Representation of a regulation cassette of a gene capableof stimulating the production of branched fatty acids.

[0078]FIG. 6: Construction scheme for the regulation cassette of the cfagene.

[0079]FIG. 7: Construction of the vector pTDEcfa

[0080]FIG. 8: Transformation of foliar disks and regeneration of theplants.

[0081] A: agrobacteria-foliar disks coculture; B: 2nd week of culture;C: 5th week of culture; D: root system of the buds; E: plantlet cuttingin vitro.

[0082]FIG. 9: Representation of a recombinant nucleic acid comprising agene coding for malonyl-CoA decarboxylase.

EXAMPLE A Construction of a Genetically Modified Plant Cell Capable ofProducing Branched Fatty Acids by Post-Elongation Alkylation

[0083] This example illustrates the construction of genetically modifiedplants capable of producing branched fatty acids by post-elongationmethylation. In particular this example describes the insertion into thegenome of the plant by genetic engineering of a gene or a set of genescoding for:

[0084] on the one hand, one or more transferases, for example,methylases or methyltransferases (of the cyclopropane fatty acidsynthase type i.e. CFA synthase, for example) capable of catalyzing theaddition of methyl groups (or other alkyl groups: ethyl, propyl,isopropyl . . . ) to the double bonds of unsaturated fatty acids; annd,

[0085] on the other, one or more genes leading to an increase in thepool of methyl donor groups (S-adenosyl methionine i.e. SAM), inparticular of the gene coding for S-adenosyl methionine synthetase (SAMsynthetase).

[0086] The construction of these transgenic plants according to theinvention is achieved according to the following steps.

[0087] Step 1: Plant Material Used.

[0088] The tobacco model plants of the SRI type (Horsch et al., 1985Science, 22, 1229-1231) or BY2 type (Nagata et al., 1992 Int.Rev. ofCytol., 132, 1-30) are used for the initial experiments. In fact, thesetwo model species can be easily transformed by Agrobacterium tumefaciens(Van Lisjsebettenns et al., 1986, J. Mol. Biol. 188, 129-145; Shaul etal. 1986; Shaul et al., 1996 Proc. Natl. Acad. Sci. USA, 93, 4868-4872)and permit the regeneration of novel plants. All oleaginous plants suchas colza, sunflower, peanut, soya, maize, etc may naturally be used inthe same manner.

[0089] Step 2: Genic Constructions

[0090] The genetic transformation consists of introducing a recombinantnucleic acid comprising an expression cassette including the gene to betransferred, the expression promoter for this gene which will be able topermit constitutive, spatial or temporal expression, and a 3′transcription termination region. In what follows the various geneticelements used and the construction of the corresponding recombinantnucleic acids are described (FIGS. 2 and 3).

[0091] 2.1. Selection and Isolation of the Nucleic Acids Coding for theEnzymes.

[0092] In this example use is made of nucleic acids coding formethylases or methyltransferases derived from prokaryotic or eukaryoticorganisms and the coding sequences of which are analogues of the codingsequence of the cyclopropane fatty acid synthase described by Wang etal. (1992 Biochemistry, 31, 11020-11028). More particularly, a nucleicacid coding for cyclopropane fatty acid synthase is isolated and itssequence verified.

[0093] Moreover, to improve the efficacy of the process, in a particularembodiment, a second nucleic acid may be used coding for the SAMsynthetase whose nucleotide sequence has been described by Peleman etal. (1989 Plant Cell, 1, 81-93).

[0094] 2.2. Selection of the Promoter Regions

[0095] For the construction of the recombinant nucleic acids, differenttypes of promoters may be used and, in particular, any promoterfunctional in plants and permitting the regulation of gene expression.

[0096] In this respect, mention may be made of the promoters permittinglocalized expression in the seeds (or certain seed tissues) of plantssuch as in particullar the promoter of the gene coding for prolamine(Zhou et al., Transgenic Res. 2 (1993) 141), the promoter of the genecoding for the pea lectin (Pater et al., Plant J. 6 (1994) 133), thepromoter of the gene coding for the LEA (“Late Embryogenesis Abundantprotein”) (Goupil et al., Plant Mol. Biol. 18 (1992) 1049), the promoterof the gene coding for the family of the napin proteins (NAP) (Boutilieret al., Plant Mol. Biol. 26 (1994) 1711), the promoter of the genecoding for rice gluterin (Zhao et al., Plant Mol. Biol. 25 (1994) 429),the promoter of the gene coding for oleosin (Keddie et al., Plant Mol.Biol. 19 (1992) 443), the promoter of the gene coding for the S familyof storage proteins (2S promoter of the napA gene, 11S or 12S promoterof the globulin gene), the promoter of the gene coding forbeta-phaseolin, legumin, gamma conglutin, concanavalin A, desaturaseBN10 (Plant Physiol. 104, 1167), wheat alpha/beta gliandin, ricecatalase CatA, sorgo alphakafirin or also maize Adh 1 (Kyozuka et al.,Plant Cell 6 (1994) 799) or pea SBP65 protein (Dehaye et al., Plant Mol.Biol. 65 (1997) 605).

[0097] In this example the promoters used are the 35S promoter ofcauliflower mosaic virus and the promoter of the nopaline synthase (nos)gene. The structure of these promoters has been described for example byBenfey and Chua (1990, Science, 250, 959-966).

[0098] 2.3. Selection of the Transcription Termination Regions.

[0099] In this example, the transcription terminating regions used arederived either directly from the gene used (FIG. 3) or from the genesfor nopaline synthase (nos) and octopine synthase (ocs).

[0100] 2.4. Construction of the recombinant nucleic acids (vectors).

[0101] In order to construct the recombinant nucleic acids enabling theabove elements to be introduced into plant cells, these genetic elementsare first isolated and subcloned in cloning vectors of the pBR322 andpUC types which bear markers permitting the selection of thetransformants which become resistant to cytotoxic agents such asantibiotics, toxins, etc. The expression cassettes are then introducedinto binary vectors containing the sequences necessary for thetransformation of the plants, in particular the T-DNA vector. Thisvector is used for the transformation of the plants and the structure ofthe vectors containing the specific recognition sequences (RB and LB)has been widely described in the literature (Book by Hoekema, the BinaryPlant Vector Systems, Offsetdrukkerij Kanters B. V., Alblasserdam, 1985.Molecular Genetics of the Bacteria-Plant Interaction, Puhler A., Ed.,Sprinnger-Verlag, NY, 1983. Plant Genetic Transformation and GeneExpression: A Laboratory Manual Draper J., Scott R., Armitage P. andWalden R. Eds, Blackwell Scientific Publications, Oxford, 1988. Kahl G.and Weising K. (1993) Genetic Engineering of Plant Cells. InBiotechnology: Genetics Fundamentals and Genetic Engineering. Rehm H.J., Reed G., Pühler A and Stadler P. Eds, VCH Publishers Inc., New York(USA) vol.2, pp 547-659).

[0102] The structure of the recombinant nucleic acids thus prepared isshown in FIGS. 2 and 3.

[0103] In another particular example the binary vector pTDEcfa wasconstructed as follows.

[0104] a) Clonage and characterization of the cfa gene coding for thecyclopropane fatty acid synthase.

[0105] On reading the article by Wang (Wang et al., 1992) an ambiguitywas noted concerning the size of the ClaI fragment present in theplasmid pAYW19. Furthermore, in the sequencing strategy Wang et al.(1992) present the ClaI fragment with a unique restriction site for theenzyme HindII upstream from the ClaI site. Now, according to thepublished sequence this site appears downstream from the ClaI site.Hence, as a first step, verification of the integrity andcharacterization of the entire structure of the cfa gene wereundertaken. For this purpose, a restriction map of the cfa gene presentin the plasmid pAYW19 was constructed with different restrictionenzymes. The analysis of the sizes of the fragments obtained afterrestriction, agarose gel electrophoresis and staining of the fragmentswith ethidium bromide made it possible to draw the physical map of thisgene, which is shown in FIG. 4. These results were confirmed bysequencing of the fragment downstream from the HindII site. The completeand corrected sequence of the cfa gene was computerized and introducedinto a software program (DNA strider) for study of the restrictionsites.

[0106] b) Construction of the Binary Vector pTDEcfa

[0107] This example describes the construction of the binary vectorpTDEcfa bearing the expression cassette of the corrected cfa gene,flanked by the LB and RB regions of the plasmid Ti. This binary vectormakes possible the transfer, by A tumefaciens, of said cassette into thegenome of the plant cells.

[0108] i) Construction of the Plasmid pBSgus

[0109] The plasmid pBSgus was constructed by cloning the EcoRI-HindIIIfragment derived from the binary vector pTDE4 in the vector pBS(pBluescript, BRL) which only contains very few restriction sites. TheEcoRI-HindII fragment contains the p35S promoter and the 3′nosterminator. Thus the vector formed, pBSgus, contains the uniquerestriction sites for the enzymes Ncol and Nrul. These two enzymes allowthe gus gene to be isolated while maintaining the integrity of theregulatory sequences (p35S and 3′nos).

[0110] ii) Creation of the Expression Cassette of the Corrected cfaGene.

[0111] The principle of this step is based on the replacement of the gusgene of the plasmid pBSgus by a cloning site comprising unique sites.This cassette was created by PCR starting from pBSgus and contains thep35S promoter and the 3′nos terminator. The primers necessary for thePCR were selected so as to create the structure shown in FIG. 5. Thechoice of the two unique restriction sites is determined by the need forthem to be absent from p35S, 3′nos, the cloning vector into which thestructure will be integrated and from the cfa gene which will beintegrated between the 2 regulatory sequences. After analysis of thedifferent sequences, two possible sites were identified: Nhel and Nrul.The construction of the cassette was achieved as follows (FIG. 6):

[0112] Cloning of the regulatory cassette in the pGEM-T in order toobtain the vector pGEM(35S-3′nos)

[0113] Creation by PCR of the Nhel site upstream and the Nrul sitedownstream from the cfa gene.

[0114] Cloning of the PCR product in the vector pGEM-T in order toobtain the vector pGEMcfa

[0115] Sequencing of the vectors pGEM(35S-3′nos) and pGEMcfa.

[0116] Cloning of the Nhel-Nrul fragment derived from the vector pGEMcfain the vector pGEM(35S-3′nos) from which the Nhel-Nrul fragment has beeneliminated. The vector obtained by this cloning is designatedpEM(35S-cfa-3′nos).

[0117] Starting from the vector pGEM(35S-cfa-3′nos) it is possible toisolate a unique EcoRI-HindII fragment containing the sequence(35S-cfa-3′nos).

[0118] After optimization of the PCR conditions for the creation of theregulatory cassette (FIG. 6), the latter was cloned in the vectorpGEM-T. Thus, after analysis by restriction with different enzymes thevector pGEM(35S-3′nos) is available in two clones.

[0119] Similarly, the vector pGEMcfa was obtained and two clones wereselected.

[0120] The desired sequence of the vectors pGEM(35S-3′nos) and pGEMcfawas computerized, then introduced into the software program DNAstrider.Two clones for each vector were sent by express mail to the Genomecompany to be sequenced.

[0121] The results of the sequencing were obtained for the two clones (2and 3) theoretically containing the vector pGEMcfa. After alignment withthe theoretical sequence, the sequence of the vector contained in clone2 exhibits several mutations and hence it will not be possible to usethis clone for further study. The sequence of the vector contained inclone 3 contains a mutation, however this mutation is a so-called“silent” mutation because it will have no effect on the sequence of theCFA protein.

[0122] The results concerning clone 3 containing the vectorpGEM(35S-3′nos) are not very encouraging because the vector contained inthis clone exhibits several mutations. However, these mutations arerelatively remote from the sequences essential for the correctfunctioning of the p35S promoter and the 3′nos terminator. Moreover,clone 4 could not be sequenced. A new selection was thus made from theclones containing the vector pGEM(35S-3′nos) and clone 10 was selectedfor sequence analysis.

[0123] Thus, the vector pGEM(35S-3′nos) contained in clone 10 wassequenced. The method of sequencing by PCR was used with 3 primers (T7,SP6 and a primer close to the poly CCC sequence). The sequence obtainedwas compared to the theoretical sequence. The 2 errors detected justbefore the HindIII site seem to be errors in the theoretical sequenceand not in the sequencing, since they are found in the sequences of thevectors pGEM(35S-3′nos) contained in clones 3 and 4 sequencedpreviously.

[0124] On the basis of the sequence results, the Nhel-Nrul fragmentderived from the vector pGEMcfa contained in clone 3 was cloned in thevector pGEM(35S-3′nos) contained in clone 10 at the Nhel and Nrul sites.The vector obtained was designated pGEM(35S-cfa-3′nos). Among the clonesobtained a selection was made using several restriction enzymes. Weselected clone 13 for further cloning.

[0125] iii) Construction of the Binary Vector

[0126] The binary vector is a vector comprising the LB and RB sequencesof the plasmid Ti, necessary for gene transfer into plant cells byAgrobacterium tumefaciens. The binary vector pTDEcfa was constructed asfollows (FIG. 7):

[0127] Cloning of the HindIII-EcoRI fragment derived from the vectorpGEM(35S-cfa-3′nos)13 into the binary vector ptde4 from which theHindIII-EcoRI fragment has been removed. The vector obtained is calledpTDEcfa. A selection was made among the clones obtained by using severalrestriction enzymes. We selected clone 8 to perform the triparentalrecombination

[0128] The clone containing the vector pTDEcfa (clone 8) was used toprepare a quantity of DNA which was tested in a Southern blotexperiment. In this experiment, the probe revealing the presence of thecfa gene by DNA-DNA hybridization was generated from the initial pAYW19vector. As a result of this experiment we were able to verify by usingdifferent restriction enzymes that the cfa gene was indeed integrated inthe binary vector between the 2 regulatory sequences: promoter andterminator.

[0129] The binary vector was then transferred to the bacterium A.tummefaciens, under the standard conditions.

[0130] Step 3: Transformation of the Plant

[0131] In a first phase, protoplasts, plant cells, tissues or tobaccoplants are genetically transformed by the recombinant nucleic acidsdescribed above by the use of indirect transfer methods with the aid ofa vector such as the agrobacteria (for example Agrobacteriumtumefaciens: C58C1Rif: pGV2260; Deblaere et al., 1985) or by usingdirect transformation methods (electroporation, particle bombardment,infiltration under pressure, etc.,(Kahl and Weising, 1993).

[0132] In the case where indirect gene transfer is used, theagrobacteria contain a plasmid possessing the vir regions (disarmed Tiplasmid) necessary for the transfer of the T-DNA into plant cells.

[0133] In a first optional step the cells are transformed by arecombinant nucleic acid (FIG. 3) coding for SAM synthetase. Thetransformants possessing a high methylating potential are then selected.

[0134] These lines or still untransformed cells are then geneticallytransformed in order to insert into their genome a recombinant nucleicaciid containing the gene coding for the CFA synthase describedpreviously.

[0135] The protocol used to perform these transformations has beendescribed by Van Lijsebettens et al.(1986 J. Mol. Biol., 188, 129-145)or itemized in the literature (Plant Genetic Transformatioon and GeneExpppression: A Laboratoryy Manual. Draper J., Scott R., Armitage P. andWalden R. Eds, Blackwell Scientific Publications, Oxford, 1988. Kahl G.and Weising K. (1993) Genetic Engineering of Plant Cells. InBiotechnology: Genetics Fundamentals and Genetic Engineering. Rehm H.J., Reed G., Pühler A and Stadler P. Eds, VCH Publishers Inc., New York(USA) vol.2, pp 547-659).

[0136] In a particular example, the undifferentiated cells of tobaccovariety BY2 were used for genetic transformation. These cells aremaintained in vitro in liquid nutritive medium. Every 10 days, a 10 mlsample of cells is taken to inoculate 100 ml of medium in a 500 mlErlenmeyer. The Erlenmeyer is placed in an orbital shaker at a speed of140 rpm in the dark at 24° C. The transformation was performed on 3 daysold cells. Two ml of cells are taken and inoculated with 100 μl of aculture of 24 h old transforming agrobacteria. The optical density atλ=600 nm of the agrobacterial culture used is adjusted to 1.5 units. Thecoculture of agrobacteria/plant cells is grown in small Petri dishes for2 days at 24° C. and in the dark. After coculture, the plant cells arerinsed abundantly with nutritive medium in order to remove as many ofthe agrobacteria as possible. The plant cells are then placed in cultureon a solid (gelified) medium. This medium contains cefotaxime toeliminate the remainder of the bacteria. It also contains kanamycin topermit the selection of the plant cells which have inserted the LB-RBfragment of the binary vector into their genome and which express thegene conferring resistance to kanamycin.

[0137] After 2 to 3 weeks of culture the plant cells resistant tokanamycin have developed. At this stage a sufficient quantity of biomassmay be taken and placed in culture in liquid medium in order toaccelerate the production of biomass, while maintaining the selectionpressure (kanamycin-cefotaxime). After several cycles of culture weremoved the selection pressure in the culture medium on a part of thebiomass in order to be able to compare the lipid profiles of thissuspension with those obtained from control cells cultured under thesame conditions, without selection pressure.

[0138] By means of this method we have thus been able to obtain a cellsuspension resistant to kanamycin. The presence of the gene coding forthe CFA in the genetic material of this suspension was also confirmed.In order to do that, we performed extractions of DNA (Dellaporta et al.,Plant Mol. Biol. Rep. 1 (1983) 19) followed by a PCR test using specificprimers for the cfa gene. The positive result of the PCR shows thepresence of a band of expected size corresponding to the cfa gene.

[0139] In another particular example, 0.5 cm diameter foliar disks wereprepared from tobacco variety SR1 cultured in vitro and transformed. Inbrief, the disks were cultured for 24 h on a solid (gelified) celldedifferentiation medium. They were then inoculated by immersion in aculture of 24 h old transforming agrobacteria, the optical density ofwhich at λ=600 nmm was adjusted to 1 unit.

[0140] Step 4: Culture and Selection of the Transformed Plants

[0141] The plant cells transformed by the vectors containing theconstructions previously described are then regenerated from isolatedcells, calluses or foliar disks by using the procedures usually used forplant cultures (Plant Cell Culture: A Practical Approach. Dixon R. A.ed., IRL Press, Oxford, Washington DC., 1985). The transformed cells andplants are placed in culture according to the usual procedures describedin the literature. The transformed cells and plants are selected whilebeing grown on a selective medium containing an antibiotic or toxinspecific for the cassette introduced.

[0142] In a particular example, the foliar disks described above weresponged on sterile blotting paper, then placed in coculture for 2 daysat 24° C. in the dark on the same solid cellular dedifferentiationmedium. After coculture, the disks were rinsed for 0.5 hour in the budinduction medium, containing cefotaxime and kanamycin. Finally, theinduction of the buds was accomplished on this same solid medium. Everyweek the disks were sampled, rinsed and replaced in culture on the samemedium prepared extemporaneously.

[0143] After 4 to 5 weeks, the buds which develop are collected andplaced in culture on a rooting medium containing cefotaxime andkanamycin.

[0144] The different steps of this transformation are illustrated inFIG. 8.

[0145] About fifty buds were obtained by this method, about twenty ofwhich had taken root on the selective medium. These plantlets arepropagated by cuttings in order to produce sufficient biomass to conductthe PCR verifications which demonstrate the presence of the cfa gene andto extract the total lipids.

[0146] Step 5: Analysis of the Transformants

[0147] The transformants thus produced are then analyzed at severallevels:

[0148] a) Direct analysis confirming the presence of the gene in theplant cell or the plant, performed by either Southern blot or PCRanalyses requiring extractions of genomic DNA (Protocol described inSambrook and/or the Current Protocol).

[0149] Sambrook J., Fritsch E. F. and Maniatis T. in Molecular Cloning.A Laboratory Manual. Irwin N., Ford N., Nolan C. and Ferguson M. eds,Cold Spring Harbor Laboratory Press 1989.

[0150] Current protocols in Molecular Biology. Ausubel F. M., Brent R.,Kingston R. E., Moore D. D., Seidman J. G., Smith J. A. and Struhl Keds, Current Protocols Inc Willey, Massachusetts . . . , vol 1:Molecular Biology—Techniques. 1994 and vol. 2: MolecularBiology—Laboratory. 1994.

[0151] b)-Analysis of the gene product i.e. mRNA by Northern blot,confirming the transcription of the gene (Protocol described in Sambrookand/or the Current Protocol cited above)

[0152] c)-Analysis of the protein synthesized, i.e. the enzyme(confirming the translation of the gene into protein) and providing acheck on the functionality of the latter (immunoblot enzymatic assay,Western blot).

[0153] The assay of SAM synthetase annd of the SAM pool in the cells isperformed more particularly by following the protocol described byMathur and Sachar (1981 FEBS Lett., 287, 113-117).

[0154] The assay of the CFA synthase in tobacco cells and plants isperformed according to the protocol described by Taylor and Cronan (1979Biochemistry, 18, 3292-3300).

[0155] d) The analysis and the identification of the triglyceridessynthesized are conducted by using chromatographic procedures of theHPLC and GC-MS type or by NMR after extraction by following theprotocols described in the literature.

[0156] These experiments permit verification that the recombinantnucleic acids are functional, that they may be introduced into plantcells, that said cells may be regenerated and that these cells actuallyproduce branched fatty acids. These acids may then be recovered directlyin the seeds of the corresponding plants by known extraction techniques.

[0157] In a particular example, the lipids were extracted from theplants obtained above (seeds, leaves or cell suspensions) by using theprocedure described by Browse et al. (Anal. Biochem. 152 (1986) 141).The lipids obtained were transesterified by the method of Fisher andCherry (Lipids 18 (1983) 589). This method uses 20% tetramethyl ammoniumhydroxide in methanol. It is nondestructive, efficient and lends itselfperfectly to the methanolysis of unusual oils. The fatty acid methylesters thus obtained were analyzed by gas chromatography.

[0158] The lipid analyses were first performed on extracts oftransformed and untransformed BV2 cell suspensions, both types beingcultured for 7 days under the same conditions (medium without selectionpressure, shaking at 140 rpm, 24° C., in the dark). These analyses wereused to validate the method of extraction of the total lipids in thecell suspensions.

[0159] Subsequently, the comparison of the lipid extracts obtained fromBY2 cell suspensions and from SR1 tobacco leaves transformed anduntransformed under different conditions (with or without selectionpressure, in the presence or absence of light, 7 or 14 days old)demonstrates the presence of compounds having the structure of branchedfatty acids according to the invention in the transformed plantmaterial.

[0160] The mass spectrometric analysis of the samples obtained confirmsthat the fatty acids obtained indeed include branched fatty acids in thesense of the invention, i.e. possessing at least one non-cyclicsubstitution. These results show in particular the presence of 9 (and/or10)-methyl octadecanoic acid and/or 9 (and/or 10)-methyl hexadecanoicacid.

[0161] Step 6: Treatment of the Branched Fatty Acids

[0162] In an optional step, the branched fatty acids produced by theplant materials of the invention are treated so as to improve theirphysicochemical properties, in particular their lubricant properties.

[0163] In a particular example, this treatmentt comprises thehydrogenation of the fatty acids in order to increase the proportion ofbranched fatty acids and thus to obtain compositions having a enhancedresistance to oxidation and low pour points. This hydrogenation isperformed under conditions known to the specialist skilled in the art.

EXAMPLE B Construction of a Genetically Modified Plant Cell Capable ofProducing Branched (Multibranched) Fatty Acids During Elongation

[0164] This example illustrates the construction of genetically modifiedplants capable of producing multibranched fatty acids. In particular,this example describes the insertion into the genome of the plant bygenetic engineering a gene or a set of genes leading the plant to use anon-linear acyl-CoA with more than 3 carbon atoms (methylmalonyl CoA forexample) instead of malonyl CoA in order to obtain branched fatty acidscalled multibranched fatty acids such as 2, 4, 6, 8-tetramethyl decanoicacid. Since the synthesis of the fatty acids occurs in the chloroplasts,the recombinant nucleic acid inserted advantageously comprises a genecoding for a malonyl-CoA decarboxylase (E.C. No. 4.1.1.9.) such thatthis enzyme is active in the chloroplast or at another level of thecell. This obtained either by introduction of the recombinant nucleicacid into the chloroplasts or by insertion of a transit peptidepermitting the address of the protein in this compartment.

[0165] The construction of these transgenic plants according to theinvention is achieved according to the following steps (FIG. 9).

[0166] Step 1: Plant Material Used

[0167] The plant material used is identical with that described inexample A.

[0168] Step 2: Genic Constructions

[0169] The genetic transformation consists of introducing an expressioncassette comprising the gene to be transferred, the promoter permittingexpression of this gene which may be constitutive, spatial or temporaland an additional nucleotide sequence coding for a transit peptideallowing the protein to be addressed to a cell compartment where it willbe active.

[0170] 2.1. Selection and Isolation of the Nucleic Acids Coding for theEnzymes

[0171] Use of the gene coding for malonyl-CoA decarboxylase whosenucleotide sequence has been described by Kolattukudy et al. (1987) orall other analogous nucleotide sequences derived from other prokaryoticor eukaryotic organisms or micro-organisms.

[0172] Kolattukudy P. E., Rogers L. M., Poulose A. J., Jang S. H., KimY. S., Cheesbrough T. M. and Liggitt D. H. (1987) Developmental patternof the expression of malonyl-CoA decarboxylase gene and the productionof unique lipids in the goose uropygial glands. Arch. Biochem. Biophys.,256, 446-454.

[0173] Fernandese N. D. and Kolattukudy P. E. (1996) Cloning, sequencingand characterization of a fatty acid synthase-encoding gene fromMycobacterium tuberculosis var. bovis BCG. Gene, 170, 95-99.

[0174] In certain cases, components of fatty acid synthesis arelocalized in the chloroplasts or other organelles. In our case, theenzyme synthesized malonyl-CoA decarboxylase, must be transported intothe chloroplasts to be active. It will be necessary to add to our genicconstructions a DNA sequence coding for an operational transit peptiderecognized by the receptor plant. These transit peptides are usuallyleader sequences which will need to be combined with the nucleotidesequence of the gene associated with a specific promoter. These “signal”sequences may be represented by any other nucleotide sequences derivedfrom that of a gene which is expressed in the cytoplasm before reachingits appropriate reaction compartment. The DNA for these transit peptidesmay be derived from other plants. It may be, for example, the transitpeptide of the small subunit of the RUBISCO of soya or the sequencescoding for the 6 or 12 terminal amino acids of the mature small subunitof the RUBISCO of pea and cited in the patent by Calgene WO 94/29467(schema of type 4).

[0175] 2.2. Selection of the Promoter Regions

[0176] In this example, the promoters used are the 35S promoter of thecauliflower mosaic virus and the promoter of the gene for nopalinesynthase (nos). The structure of these promoters has been described forexample by Benfey and Chua (1990, Science, 250, 959-966).

[0177] 2.3. Selection of the transcription terminating regions

[0178] In this example, the transcription terminating regions used arederived from the genes for nopaline synthase (nos) and octopine synthase(ocs) (FIG. 9).

[0179] 2.4. Construction of the Recombinant Nucleic Acids (Vectors)

[0180] In order to construct the recombinant nucleic acids which enablethe above elements to be introduced into the plant cells, these geneticelements are first isolated and subcloned into pBR322 and pUC typecloning vectors, which bear markers permitting the selection of thetransformants which acquire resistance to cytotoxic agents such asantibiotics, toxins, etc. The expression cassettes are then introducedinto binary vectors containing the sequences necessary for thetransformation of plants, in particular, the T-DNA vector. This vectoris used for the transformation of the plants and the structure of thevectors containing the specific recognition sequences RB and LB) hasbeen extensively described in the literature. (Book by Hoekema, theBinary Plant Vector Systems, Offsetdrukkerij Kanters B. V.,Alblasserdam, 1985. Molecular Genetics of the Bacteria-PlantInteraction, Puhler A., Ed., Sprinnger-Verlag, N.Y., 1983. Plant GeneticTransformation and Gene Expression: A Laboratory Manual Draper J., ScottR., Armitage P. and Walden R. Eds, Blackwell Scientific Publications,Oxford, 1988. Kahl G. and Weising K. (1993) Genetic Engineering of PlantCells. In Biotechnology: Genetics Fundamentals and Genetic Engineering.Rehm H. J., Reed G., Pühler A and Stadler P. Eds, VCH Publishers Inc.,New York (USA) vol.2, pp 547-659).

[0181] The structure of the recombinant nucleic acids thus prepared isshown in FIG. 9.

[0182] Step 3: Transformation of the Plant

[0183] In a first phase, protoplasts, plant cells, tobacco plants ortissues thereof are genetically transformed by the recombinant nucleicacids described above by use of indirect transfer methods using a vectorsuch as the agrobacteria (for example, Agrobacterium tumefaciens:C58C1Rif : pGV2260; Deblaere et al., 1985) or by using directtransformation methods (electroporation, particle bombardment,infiltration under vacuum, etc., (Kahl and Weising, 1993).

[0184] In the case of the indirect gene transfer, the agrobacteriacontain a plasmid possessing the vir regions (disarmed Ti plasmid)necessary for the transfer of the T-DNA into the plant cells.

[0185] The cells are genetically transformed in order to insert intotheir genome a recombinant nucleic acid (FIG. 9) containing the genecoding for the malonyl-CoA decarboxylase previously described.

[0186] The protocol used to perform these transformations has beendescribed by Van Lijsebettens et al. (1986 J. Mol. Biol., 188, 129-145)and itemized in the literature. (Plant Genetic Transformation and GeneExpression: A Laboratory Manual Draper J., Scott R., Armitage P. andWalden R. Eds, Blackwell Scientific Publications, Oxford, 1988. Kahl G.and Weising K. (1993) Genetic Engineering of Plant Cells. InBiotechnology: Genetics Fundamentals and Genetic Engineering. Rehm H.J., Reed G., Pühler A and Stadler P. Eds, VCH Publishers Inc., New York(USA) vol.2, pp 547-659).

[0187] Step 4: Culture and Selection of the Transformed Plants

[0188] The plant cells transformed by the vectors containing theconstructions previously described are then regenerated from isolatedcells, calluses or foliar disks by using the usual procedures for plantcultures (Pllant Cell Culture: A Practical Approach. Dixon R. A. ed.,IRL Press, Oxford, Wash. D.C., 1985). The transformed cells and plantsare placed in culture in accordance with the usual procedures describedin the literature. The transformed cells and plants are selected duringtheir culture on a selective medium containing an antibiotic or toxinspecific for the cassette introduced.

[0189] Step 5: Analysis of the Transformants

[0190] The transformants thus produced are then analyzed at severallevels:

[0191] a) Direct analysis confirming the presence of the gene in theplant cell or the plant, performed by either Southern blot or PCRanalyses requiring extractions of genomic DNA according to a Protocoldescribed in

[0192] Sambrook J., Fritsch E. F. and Maniatis T. in Molecular Cloning.A Laboratory Manual. Irwin N., Ford N., Nolan C. and Ferguson M. eds,Cold Spring Harbor Laboratory Press 1989 or,

[0193] Current protocols in Molecular Biology. Ausubel F. M., Brent R.,Kingston R. E., Moore D. D., Seidman J. G., Smith J. A. and Struhl Keds, Current Protocols Inc Wiley, Massachusetts . . . , vol 1: MolecularBiology—Techniques. 1994 and vol. 2: Molecular Biology—Laboratory. 1994.

[0194] b) Analysis of the gene product i.e. mRNA by Northern blot,confirming the transcription of the gene (Protocol described in Sambrookand/or the Current Protocol cited above)

[0195] c) Analysis of the protein synthesized, i.e. the enzyme(confirming the translation of the gene into protein) and providing acheck on the functionality of the latter (immunoblot enzymatic assay,Western blot).

[0196] d) The assay of malonyl-CoA decarboxylase according to theprotocol described by Kolattukudy et al. (1987): Developmental patternof the expression of malonyl-CoA decarboxylase gene and the productionof unique lipids in the goose uropygial glands. Arch. Biochem. Biophys.,256, 446-454.

[0197] e) The analysis and the identification of the triglyceridessynthesized are conducted by using chromatographic procedures of theHPLC and GC-MS type or by NMR after extraction by following theprotocols described in the literature.

[0198] These experiments permit verification that the recombinantnucleic acids are functional, that they may be introduced into plantcells, that said cells may be regenerated and that these cells actuallyproduce branched fatty acids. These acids may then be recovered directlyin the seeds of the corresponding plants by known extraction techniques.

1. Production process for branched fatty acids, characterized in thatsaid branched fatty acids are produced from at least one plant cell orfrom one plant material or from a plant comprising at least one plantcell, said plant cell comprising in its genome a recombinant nucleicacid coding for a product which induces or stimulates the synthesis ofbranched fatty acid(s).
 2. Process according to claim 1, characterizedin that it comprises in addition an extraction step of the branchedfatty acids.
 3. Process according to claim 2, characterized in that itcomprises in addition a treatment step of the extracted branched fattyacids.
 4. Process according to one of the claims 1 to 3, characterizedin that the recombinant nucleic acid codes for a product which inducesor stimulates the postsynthetic branching of the fatty acids produced bysaid cell.
 5. Process according to claim 4, characterized in that therecombinant nucleic acid codes for an enzyme permitting the transfer ofone or more alkyl groups to the double bond(s) of the unsaturated fattyacids.
 6. Process according to claim 5, characterized in that therecombinant nucleic acid codes for a methyl transferase.
 7. Processaccording to claim 5, characterized in that the recombinant nucleic acidcodes for a cyclopropane fatty acid synthase.
 8. Process according toone of the claims 4 to 7, characterized in that the plant cell comprisesin addition a recombinant nucleic acid coding for SAM synthetase. 9.Process according to one of the claims 1 to 3, characterized in that therecombinant nucleic acid codes for an enzyme which forces said plantcell to use a substrate comprising more than 3 carbon atoms for thesynthesis of the aliphatic chain.
 10. Process according to claim 9,characterized in that the recombinant nucleic acid codes for an enzymecapable of forcing the plant to use a non-linear acyl-CoA, such as inparticular methylmalonyl CoA.
 11. Process according to claim 10,characterized in that the recombinant nucleic acid codes for a malonylCoA decarboxylase.
 12. Recombinant nucleic acid characterized in that itcomprises: a nucleic acid coding for a product which induces orstimulates the synthesis of branched fatty acid(s), a promoterregulating the expression of said nucleic acid and capable of causingthe localized expression of this nucleic acid in certain plant tissuesor certain plant parts, and, a 3′ transcription termination region. 13.Nucleic acid according to claim 12, characterized in that the promoteris a promoter capable of causing localized expression of the nucleicacid in the seed of a plant.
 14. Recombinant nucleic acid comprising: anucleic acid coding for a methyl transferase capable of catalyzing thetransfer of a methyl group to an aliphatic chain of an unsaturated fattyacid, a functional promoter in the plant cells regulating the expressionof said nucleic acid, and a 3′ transcription termination region. 15.Recombinant nucleic acid comprising: a nucleic acid coding for an enzymewhich forces a plant cell to use a substrate comprising more than 3carbon atoms as substrate for the synthesis of the aliphatic chain, inparticular for a malonyl CoA decarboxylase, a functional promoter in theplant cells regulating the expression of said nucleic acid, and a 3′transcription termination region.
 16. Recombinant nucleic acid accordingto any one of the claims 12 to 15, characterized in that its sequencecomprises in addition a nucleic acid coding for the SAM synthetase. 17.Vector comprising a recombinant nucleic acid according to any one of theclaims 12 to
 16. 18. Plant cell comprising a recombinant nucleic acidsuch as defined in one of the claims 12 to 16 or a vector according toclaim
 17. 19. Plant cell according to claim 18, characterized in that itis an oleaginous plant cell, preferably selected from colza, sunflower,peanut, soya, flax and maize.
 20. Transgenic plant characterized in thatit contains at least one cell according to claim 18 or
 19. 21.Transgenic plant characterized in that it contains in at least one partat least of its cells, a nucleic acid according to the claims 12 to 16or a vector according to claim
 17. 22. Production process for branchedfatty acids by cell culture of a plant cell according to either of theclaims 18 or 19 comprising: the culture of these cells in a mediumsuitable for their growth, the extraction and purification of thebranched fatty acids from the cells or from the supernatant of saidculture.
 23. Preparation process for branched fatty acids from atransgenic plant whose cells contain a recombinant nucleic acidaccording to any one of the claims 12 to 16, characterized in that itcomprises: the field culture of said transgenic plants; the recovery ofthe seeds of said plants; the extraction of the fatty acids from theseseeds.
 24. Use of a transgenic plant according to claim 20 or 21,regenerated from a cell according to either of the claims 18 or 19, forthe production of branched fatty acids.
 25. Use of the whole or part ofa transgenic plant, at least some of the cells of which contain arecombinant nucleic acid according to claim 12 for the production ofbranched fatty acids.
 26. Branched fatty acids which can be obtained bythe process of any one of the claims 1 to 11, 22 or
 24. 27. Compositioncontaining branched fatty acids obtained by the process of any one ofthe claims 1 to 11, 22 or
 24. 28. Use of a branched fatty acid esterwhich can be obtained by the process of any one of the claims 1 to 11,22 or 23 for the preparation of a lubricant composition.
 29. Preparationprocess of a transgenic plant capable of producing branched fatty acids,comprising the introduction of a recombinant nucleic acid such asdefined in claim 12 into a plant cell or part of a plant and theregeneration of a transgenic plant from these plant cells or parts ofplants.